POS0473 INFLAMMATORY-PRIMED MUSCLE CELLS INFLUENCE MACROPHAGE CYTOKINE SECRETION IN A MYOSITIS CONTEXT
Authors: B. Mccord, R. Day
Journal: Annals of the Rheumatic Diseases (2022)

Background Sporadic inclusion body myositis (sIBM) is a progressive disease causing muscle weakness and mobility issues. It is characterised by inflammatory and degenerative features; however there is no clear single cause for sIBM symptoms. Inflammatory factors include macrophage infiltration into muscle fibres and presence of high levels of inflammatory cytokines such as IFNγ [1]. Degenerative features within muscle fibres include sarcoplasmic inclusions containing aggregated proteins. p62 and TAR DNA-binding protein 43 (TDP-43) are two proteins that are found in inclusion bodies, and these have been suggested as sensitive diagnostic markers differentiating sIBM from other inflammatory myopathies [2]. Currently there is limited understanding of the pathogenic mechanisms underlying sIBM, and no effective treatments. Objectives The aim of this study is to investigate the interplay between inflammatory and degenerative features of sIBM with a focus on macrophage secreted factors, p62 and TDP-43 sarcoplasmic aggregation. Firstly, to examine whether skeletal muscle cells that have previously been exposed to an inflammatory environment can in turn influence the inflammatory response of macrophages. Secondly, by investigating if macrophage-secreted inflammatory factors influence p62 and TDP-43 sarcoplasmic aggregates. This will give insight into whether macrophage inflammation precludes skeletal muscle degeneration in an sIBM context or vice versa. Methods Primary human myogenic cells from healthy individuals were differentiated into myotubes for 7 days, with further 48-hour 20 ng/mL IL-1β and 750 ng/mL IFNγ incubation to form inflammatory-primed myotubes. Conditioned media (CM) was collected from washed myotubes after 24 hours. 6x10 5 THP-1 cells/mL were differentiated to unprimed M(PMA) macrophages with 150 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours, followed by 72-hour PMA-free rest. Inflammatory-primed M(IFNγLPS) THP-1 were generated from M(PMA) with 48-hour IFNγ and lipopolysaccharide (LPS) incubation. 20 % CM from myotubes was added to M(PMA) or M(IFNγLPS) THP-1 for 24 hours, before washing and measuring IL-6 and TNFα release from macrophages via ELISA. 20 % CM from M(PMA) or M(IFNγLPS) was added to myotubes for 48 hours before quantifying TDP-43 and p62 aggregates with image analysis. Results M(IFNγLPS) THP-1 secreted higher levels of IL-6 and TNFα than M(PMA). M(PMA) or M(IFNγLPS) exposed to conditioned media from control or inflammatory-primed myotubes had no detectable TNFα secretion. For both M(PMA) and M(IFNγLPS), addition of inflammatory-primed myotube CM caused higher IL-6 secretion than adding control myotube CM. Incubating unprimed myotubes with M(PMA) or M(IFNγLPS) CM had no effect on the presence, size, or frequency of p62 or TDP-43 sarcoplasmic aggregates compared to control conditions. Conclusion Inflammatory priming myotubes with IL-1β and IFNγ caused a response in macrophages that increased IL-6 production. However, CM from inflammatory-primed myotubes did not influence TNFα secretion from macrophages compared to control myotube CM. IL-6 has pleiotropic effects on skeletal muscle [3], promoting both hypertrophy and atrophy under different conditions. Therefore the effects of macrophage IL-6 upregulation on muscle is unclear. Culturing myotubes in unprimed or inflammatory-primed macrophage conditioned media did not influence p62 or TDP-43 sarcoplasmic aggregation. This suggests macrophage secretory factors are not responsible for the sIBM degenerative features of p62 and TDP-43 aggregation. References [1]M. Rigolet et al. , Distinct interferon signatures stratify inflammatory and dysimmune myopathies. RMD Open 5 , e000811 (2019). [2]O. Dubourg et al. , Diagnostic value of markers of muscle degeneration in sporadic inclusion body myositis. Acta myol 30 , 103-108 (2011). [3]P. Muñoz-Cánoves et al., Interleukin-6 myokine signaling in skeletal muscle: a double-edged sword? Febs j 280 , 4131-4148 (2013). Disclosure of Interests None declared.